【作者】 霍春玲；

【导师】 陈晓玲；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:肺纤维化（pulmonary fibrosis, PF）可引起肺动脉高压（pulmonary artery hypertension, PAH）,长期PAH可导致肺心病（pneumocardial disease, PHD）,是PF致死的重要原因之一。目前PF发生过程中PAH的形成机制尚不清楚,也无有效的防治方法。有研究表明,在博莱霉素（bleomycin, BLM）诱导的PF形成过程中,肺动脉内皮细胞受损。另有研究发现,气管内滴注BLM可引起肺动脉内皮细胞内皮源性一氧化氮合酶（endothelium derived nitro oxide synthase, eNOS）mRNA表达减少,这可能是慢性PAH形成的机制之一。因此有效地保护肺动脉内皮细胞可能对于防治BLM诱导的PAH具有重要意义。罗格列酮（Rosiglitazone, RSG）属噻唑烷二酮（thiazolidines, TZDs）类,临床主要用于治疗2型糖尿病。有研究结果表明,RSG可以激活过氧化物酶体增殖活化受体-γ（peroxisome proliferator-activated receptor-γ, PPAR-γ）。PPAR-γ在血管、心脏组织均有广泛表达,其激活后可参与调节炎症、细胞凋亡、平滑肌迁移等过程。以往研究表明,血管组织中的PPAR-γ对内皮细胞具有保护作用,PPAR-γ还可通过促进内皮源性一氧化氮（nitric oxide, NO）的生成发挥降低血压的作用。RSG是否具有抗BLM诱导的PAH及内皮损伤的作用,目前尚未见报道。内皮细胞可以产生许多血管活性物质,这些物质作用于血管平滑肌,对于维持血管的张力有重要意义。在这些血管活性物质中,内皮源性的NO可以作用于血管平滑肌,舒张血管,抑制血小板聚集,在许多生理或病理状态下,对血管的功能具有重要的调节作用。凌亦凌等发现,在内毒素诱导的肺动脉高压过程中,肺动脉内皮受损,内皮源性NO生成减少,导致了肺动脉血管反应性紊乱。在BLM诱导的PAH的形成过程中,是否会引起肺动脉血管反应性紊乱,尚不清楚。因此,本实验的目的是观察RSG对BLM诱导大鼠PF过程中所致PAH的影响及BLM诱导肺动脉血管反应性的变化。材料方法和结果:本实验将采用气管滴入BLM复制大鼠PF模型;应用右心漂浮导管技术测定大鼠平均肺动脉压（mean pulmonary artery pressure, mPAP）;采用光学显微镜观察肺动脉形态学改变;应用离体血管环张力测定技术观察肺动脉血管环（pulmonary artery rings, PARs）反应性的变化。本实验分两部分进行,具体方法与实验结果见下列内容。一、罗格列酮对博莱霉素诱导大鼠肺纤维化所致肺动脉高压的影响方法:将64只雄性SD大鼠随机分为八组:①对照组（n=9）:正常大鼠;②B+NS14d组（n=10）:一次性气管滴入BLM（5mg/0.5ml/kg）后,2ml生理盐水（normal saline, NS）灌胃14天;③B+R14d组（n=12） :一次性气管滴入BLM（5mg/0.5ml/kg）后,RSG（3mg/kg）溶于2mlNS中灌胃14天;④R14d组（n=9）:一次性气管滴入NS（0.5ml/kg）后,RSG（3mg/kg）溶于2mlNS中灌胃14天;⑤B+NS28d组（n=6）:一次性气管滴入BLM（5mg/0.5ml/kg）后2mlNS灌胃28天;⑥B + R28d组（n=6） :一次性气管滴入BLM（5mg/0.5ml/kg）后,RSG（3mg/Kg）溶于2mlNS中灌胃28天;⑦R28d组（n=7）:一次性气管滴入NS（0.5mL/kg）后,RSG（3mg/kg）溶于2mlNS中灌胃28天;⑧B+R14d+N14d组（n=6）:一次性气管滴入BLM（5mg/0.5ml/kg）后,RSG（3mg/kg）溶于2mlNS中灌胃14天,之后继续2mlNS灌胃14天。各组动物分别在气管滴注NS或BLM后14,28天进行mPAP的测定。①、②、③组各有3只动物用于观察肺动脉形态学改变。结果: 1 RSG对BLM诱导的PAH的影响与对照组相比,B+NS14d组mPAP明显增高（19.00±2.94 mmHg vs. 14.83±1.17mmHg, P<0.05）,B+R14d组mPAP无明显差别（13.11±1.96mmHg vs. 14.83±1.17 mmHg, P>0.05）,B+R28d组mPAP明显降低（11.33±2.07 mmHg vs. 14.83±1.17 mmHg, P<0.05）,B+R14+N14组mPAP明显降低（11.83±1.33 mmHg vs. 14.83±1.17 mmHg,P<0.05）。与B+NS14d组相比,B+R14d组mPAP明显降低（13.11±1.96 mmHg vs. 19.00±2.94 mmHg, P<0.05）。2 RSG对BLM引起的肺动脉形态学改变的影响光镜下可见对照组血管内皮细胞排列连续,整齐,内皮下弹力纤维完整,平滑肌层结构整齐;B+NS14d组部分内皮细胞脱落,内皮下弹力纤维结构尚可;B+R14d组内皮细胞损伤较轻,内皮基本完整,内皮下弹力纤维基本完整,平滑肌结构基本整齐。小结: BLM气管滴入14天可导致大鼠肺动脉压显著升高,肺动脉内皮细胞损伤。RSG逆转了BLM诱导的肺动脉压的升高,同时减轻了肺动脉内皮细胞的损伤,提示RSG可能通过保护血管内皮细胞降低肺动脉压。二、博莱霉素诱导大鼠肺动脉高压时肺动脉血管反应性的变化方法:24只雄性SD大鼠随机分为四组（n=6）:①有内皮对照组:一次性气管滴入NS0.5ml/kg后14天取材;②无内皮对照组:一次性气管滴入NS0.5ml/kg后14天,去除肺动脉内皮;③有内皮B14d组,一次性气管滴BLM5mg/ 0.5ml/kg后14天取材;④无内皮B14d组,一次性气管滴入BLM5mg/0.5ml/kg后14天,去除肺动脉内皮。腹腔注射25%乌拉坦（0.4mg/100g）麻醉后,固定于手术台,联合取出大鼠心肺,分离并制备离体PARs。将血管环固定于血管张力测定装置的浴槽内,用10^-6mol/L苯肾上腺素（PE）预收缩PARs致反应平台,然后对有内皮组PARs测定:⑴对10^-6mol/L PE的收缩反应性;⑵对10^-6mol/L乙酰胆碱（acetyl -choline, ACh）的舒张反应性;⑶用N（omega）-nitro-L-arginine methyl ester（L-NAME, 10-4mol/L）孵育5min后,再测定各组PARs对PE(10^-6mol/L)的收缩反应性变化。在去内皮组,用PE(10^-6mol/L)预收缩PARs致反应平台后,PARs对ACh(10^-6mol/L)的舒张反应性消失,说明内皮已去除干净,然后对PARs进行测定:⑴对PE(10^-6mol/L)的收缩反应性;⑵对NO的供体硝普钠（sodium nitroprusside,SNP）的累积浓度舒张反应曲线(SNP, 10^-9～10^-6mol/L);⑶用N（omega）- Nitro -L-arginine methyl ester（L-NAME, 10-4mol/L）孵育5min后,再测定各组PARs对PE(10^-6mol/L)的收缩反应性变化。实验结束后用将血管环烘烤至重量不再变化,记录干重。收缩反应结果用每毫克血管环干重的克张力（g/mg.d.w）表示,舒张反应性结果以占PE(10^-6mol/L)收缩值的百分比表示。结果: 1 BLM滴入后PARs对PE收缩反应性的变化有内皮B14d组PARs对PE收缩反应性显著高于有内皮对照组PARs（0.73±0.17g/mg.d.w vs. 0.47±0.23g/mg.d.w, P<0.05 ）。在无内皮组B14d组PARs对PE收缩反应性也显著高于无内皮对照组PARs（0.97±0.18 g/mg.d.w vs. 0.73±0.18g/mg.d.w, P<0.05）。提示BLM可以导致肺动脉平滑肌对PE的收缩反应性增强。2内皮细胞在PARs对PE收缩反应中的作用无内皮对照组PARs对PE收缩反应性显著高于有内皮对照组PARs（0.73±0.18g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05）。无内皮B14d组PARs对PE收缩反应性也显著高于有内皮的B14d组PARs（0.97±0.18 g/mg.d.w vs. 0.73±0.17 g/mg.d.w, P<0.05）。提示内皮细胞的损伤可增强PARs对PE的收缩反应性。3 BLM对血管内皮的损伤作用有内皮B14d组PARs对ACh舒张反应性显著低于有内皮对照组PARs（34.21±13.97% vs.83.63±6.57%, P<0.01）,提示BLM损伤了血管内皮,使PARs对ACh的舒张反应性降低。4 L-NAME（10-4mol/L）孵育对PARs反应性的影响有内皮对照组PARs在L-NAME孵育后对PE的收缩反应性明显增强（0.71±0.42 g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05）。无内皮对照组PARs及有内皮B14d组PARs和无内皮B14d组PARs在L-NAME孵育前后对PE的收缩反应性无明显变化。5 PARs对SNP的累积浓度舒张反应的影响随SNP浓度的增加,无内皮对照组PARs及无内皮B14d组PARs,舒张反应性逐渐增加,但组间无显著差别。提示外源性NO的舒张肺动脉血管作用不依赖于血管内皮细胞。小结:BLM可导致PARs对PE的收缩反应性增强,对ACh的舒张反应性降低,肺动脉内皮功能障碍,内皮源性NO的减少,平滑肌功能异常,使血管反应性紊乱,这可能是大鼠肺纤维化形成初期BLM导致肺动脉压升高的重要机制之一。结论:1在大鼠肺纤维化形成初期,BLM可以引起肺动脉压升高,这可能与BLM使肺动脉内皮损伤,内皮源性NO的减少,平滑肌功能异常,血管反应性紊乱有关。2 RSG可以防治大鼠肺纤维化形成初期发生的肺动脉升高,这可能与其保护肺动脉内皮细胞有关。更多还原

【Abstract】 ObjectivePulmonary Fibrosis（PF） can induce Pulmonary artery hypertension（PAH）. Long-term PAH can induce pneumocardial disease（PHD）, which is one of the most important lethal facors of PF. Up to now, the mechanism of PAH generated in PF has not been clear. And there is still no ideal method for prevention and therapy. Studies have shown that pulmonary artery endothelial cells were injured in the formation of PF induced by BLM. Other researches have indicated that intratracheal instillation of BLM can lead to the decrease of endothelium nitric oxide synthase （eNOS） expressed in pulmonary artery endothelial cells, which may be one of the mechanisms implicated in chronic PAH. Thus the effective protection of pulmonary artery endothelial cells may play an important role in the prevention and therapy of PAH induced by BLM.Rosiglitazone （RSG）, is a kind of Thiazolidines （TZDs） drugs, was mainly used in the therapy of type II diabetes. Some researches show that RSG can activate peroxisome proliferator-activated receptor-γ（PPAR-γ）. The activation of PPAR-γ, expressed abundantly both in blood vessels and heart, participates in the modulation of inflammation, cell apoptosis, migration of vascular smooth muscles etc. Previous researches have shown that PPAR-γexpressed in blood vessels can protect the endothelial cells and induce the decrease of the blood pressure by facilitate the release of endothelium derived NO（nitric oxide）. But, whether RSG has the reversal effects on PAH and the injury of pulmonary artery endothelial cells induced by BLM has not been reported.Endothelial cells can generate many vasoactive substances, which are very important to the maintenance of the vascular tension. Among these vasoactive substances, endothelium derived NO has the effects on the relaxation of blood vessels and the inhibition of platelet aggregation. It is an important modulator of vascular reactivity under many physiologic or pathophysiologic conditions. Ling YL et al reported that the injury of pulmonary artery endothelial cells and the decrease of endothelium derived NO release, both generated in the PAH induced by lipopolysaccharide （LPS）, led to the disorder of pulmonary artery vascular activity. Howerver, whether the injury of pulmonary artery endothelial cells in the PAH induced by BLM can lead to the disorder of pulmonary artery vascular reactivity has not been clear.Thus, in our research, we observed the effects of RSG on PAH generated in PF induced by BLM and the effects of pulmonary artery endothelial cells on the changes of pulmonary artery vascular reactivity induced by BLM.Rats model of PF were duplicated by intratracheal instillation of BLM. Right Cardiac Catheter technique was applied to detect rat mean pulmonary artery pressure（mPAP）. Light microscope was used to observe the morphologic changes of pulmonary artery. The changes of PARs reactivity were observed utilizing the PARs technique.Part1 Effects of RSG on Pulmonary Artery Hypertension during Pulmonary Fibrosis Induced by BleomycinMethods:Sixty-four male Sprague-Dawley rats were randomly·divide -d into eight groups:①Control group（n=9）: Normal rats;②B + NS14d group（n=10）: BLM(5mg·0.5ml^-1·kg^-1) was administrated by single intratracheal instillation and then NS （2ml per rat） was administrated by intragastric gavage（i.g） every day of the following 14 days;③B+R14d group（n=12）: BLM was administrated by single intratracheal instillation (5mg·0.5ml^-1·kg^-1) and then RSG (3mg·kg^-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 14 days;④R14d group（n=9）: NS(0.5ml·kg^-1) was administrated by single intratracheal instillation and then RSG (3mg·kg^-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 14 days;⑤B+NS28d group（n=6）: BLM(5mg·kg^-1) was administrated by single intratracheal instillation and then NS （2ml per rat, i.g） was administrated every day of the following 28 days;⑥B+R28d group（n=6）: BLM(5mg·0.5ml^-1·kg^-1) was administrated by single intratracheal instillation and then RSG (3mg·kg^-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 28 days;⑦R28d group（n=7）: NS (0.5ml·kg^-1) was administrated by single intratracheal instillation and then RSG (3mg·kg^-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 28 days;⑧B+R14d+N14d group（n=6）: BLM (5mg·0.5ml^-1·kg^-1) was administrated by single intratracheal instillation and then RSG (3mg·kg^-1 per rat, dissolved in 2ml NS, i.g) was administrated every day of the following 14 days, followed by NS（2ml per rat, i.g） administrated every day of the following 14 days. RSG or NS was administrated 2h after BLM or NS intratracheal instillation. mPAP of each group was detected respectively 14 or 28 days after NS or BLM intratracheal instillation. Three rats in each（①,②,③） group were sacrified to observe the morphologic changes of the PARs.Results:1 Effects of RSG on PAH induced by BLM Compared with control group, the mPAP of B+NS14d group was significantly incresed（19.00±2.94 mmHg vs. 14.83±1.17mm Hg, P<0.05）, the mPAP of B+R14d group had no significant differences, while the mPAP of B+R28d group and B+R14+N14 group were decreased significantly（11.33±2.07 mmHg vs. 14.83±1.17 mmHg, P<0.05; 11.83±1.33 mmHg vs. 14.83±1.17mmHg, P<0.05）. Compared with B+NS14d group, the mPAP of B+R14d group was significantly decreased （13.11±1.96 mmHg vs. 19.00±2.94 mmHg, P<0.05）.2. Effects of RSG on morphologic changes of PARs The LM results showed, vascular endothelial cells in control group were intact and continuous, with the smooth muscle layers arranged orderly; In B+NS14d group, vascular endothelial cells apeared losing. The structure of the elastic fiber beneath the endothelium was nearly normal. The structure of smooth muscles were well arranged. Compared with Control group, no significant change was observed in B+R14 group.The results suggested:Fourteen days after intratracheal instillation, BLM induced significant increase of rats mPAP. RSG may have the reversing function to the increase of rats mPAP induced by BLM. RSG can inhibit the pulmonary artery endothelial cells injury induced by BLM, which implied that RSG may decrease mPAP by protecting pulmonary artery endothelium.Part2 Reactivites of PARs during PAH Induced by BLM Methods:Twenty-four Sprague-Dawley rats were randomly divided into four groups （n=6 in each group）:①Control with endothelium group: Normal rats;②Control without endothelium group: Normal rats. The pulmomary artery endothelium was removed;③B14d with endothelium group: BLM (5mg·0.5ml^-1·kg^-1) was administrated by single intratracheal instillation. Rats were sacrificed 14 days later;④B14d without endothelium group: BLM was administrated by single intratracheal instilled(5mg·0.5ml^-1·kg^-1). Rats were sacrificed 14 days later. The pulmonary artery endothelium was removed. Changes of vascular tension of pulmonary artery rings （PARs） were detected in vitro. PARs were carefully prepared. The contraction responses to phenylephrine(PE,10^-6mol/L) were then tested separately to observe the stability of the PARs reactivity. When the contraction responses had become stable, the group with endothelium was detected:①recording curve of the contraction responses of PARs to PE(10^-6mol/L);②recording curve of the relaxation responses of PARs to acetylcholine(ACh, 10^-6mol/L);③recording curve of the contraction responses of PARs to PE(10^-6mol/L) after preincubation with N（omega）-nitro-L-arginine methyl ester （L-NAME, 10-4mol/L） for 5 min. As to the groups without endothelium, when the contraction responses to PE(10^-6mol/L) had become stable, the relaxation responses to ACh(10^-6mol/L) were observed. The disappearance of the PARs relaxation responses to ACh(10^-6mol/L) was the identification of the complete endothelium removing. Then recorded: the group without endothelium:①recording curve of the contraction responses of PARs to PE (10^-6mol/L);②recording curve of the cumulative dose relaxation responses of PARs to sodium nitroprusside (SNP, 10^-910^-6mol/L);③recording curve of the contraction responses of PARs to PE(10^-6mol/L) after preincubation with L-NAME（10-4mol/L） for 5 min. The PARs responses to PE were expressed as g/mg.dw, and vascular relaxation responses to ACh or SNP were expressed as percentage reduction of initial vascular tension induced by PE. Results: 1 The changes of PARs contraction responses to PE after BLM intratracheal instillation Compared with the PARs in control group with endothelium, the contraction responses to PE of PARs in B14d with endothelium group were significantly increased （0.73±0.17 g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05）. Compared with the PARs in control group without endothelium, the contraction responses to PE of PARs in B14d without endothelium group were significantly increased （0.97±0.18 g/mg.d.w vs. 0.73±0.18g/mg.d.w, P<0.05）. The results indicated that BLM enhanced the contraction responses of pulmonary smooth muscles to PE.2 The contraction responses of PARs endothelial cells to PE Compared with the PARs in control group with endothelium, the contraction responses to PE of PARs in control group without endothelium were significantly increased （0.73±0.18g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05）. Compared with PARs in B14d with endothelium group, the contraction responses to PE of PARs in B14d without endothelium group were significantly increased（0.97±0.18g/ mg.d.w vs. 0.80±0.15g/ mg.d.w, P<0.05）. The results indicated that endothelial cells injury can enhance the PARs contraction responses to PE.3 The injury effects of BLM on pulmonary artery endothelial cells Compared with PARs in control group with endothelium, the relaxation responses to ACh of PARs in B14d with endothelium group were significantly decreased （34.21±13.97% vs.83.63±6.57%, P<0.01）, which indicated that BLM injured the pulmonary artery endothelium, and attenuated the relaxation responses of PARs to ACh.4 Effects of L-NAME preincubation on PARs reactivities After incubation with L-NAME, the contraction responses of PARs to PE in control with endothelium group increased significantly（0.71±0.42 g/mg.d.w vs. 0.47±0.23 g/mg.d.w, P<0.05）, while the contraction responses of PARs to PE in control without endothelium group, B14d with endothelium group and B14d without endothelium group had no significant differences.5 Effets of SNP cumulative dose on relaxation responses of PARs With the increase of SNP concentration, the relaxation responses of PARs increased gradually, but there was not significant difference between the control without endothelium group and B14d without endothelium group. The results indicated that the pulmonary artery relaxation responses to exogenous NO were independent on endothelial cells.The results suggested:BLM increased the contraction responses of PARs to PE and decreased the relaxation resonses of PARs to ACh, decreased the endothelium derived NO, made the function of endothelial cells and vascular smooth muscles abnormal, led to PARs reactivities disorder, which maybe one of the important mechanisms involved in the increase of pulmonary artery pressure induced by BLM during the initial period of PF in rats.Conclusion:1 BLM induced the increase of pulmonary artery pressure in initial period of PF in rats, which maybe have the relationship with the injured pulmonary artery endothelial cells, the decreased endothelium derived NO and the abnormal function of vascular smooth muscles that led to the PARs reactivities disorder.2 RSG can prevent and cure the increase of pulmonary artery pressure in initial period of PF in rats, which implied that RSG may decrease mPAP by protecting pulmonary artery endothelial cells.更多还原